In order to allow proteins bearing a principal role in the biological life functions to orderly and properly exhibit their functions in vivo, post-translation modifications including glycosylation play an extremely important role. With regard to the post-translation modifications, the following findings were gradually made in recent years. The majority of the proteins in a living body are modified with sugar chains, and those sugar chains attached to the proteins play important roles in various aspects of biological phenomena, including protein stability, binding with hormones, binding with toxins, viral infection, mycoplasma infection, bacterial infection, protozoan infestation, fertilization, development and differentiation, cancer cell metastasis, apoptosis and the like. Even when proteins have the same amino acid sequence and the same name, such proteins are modified with a wide variety of sugar chains and, depending on the condition of the protein-producing cells, the structures of the sugar chains vary and the proteins thus have different roles in vivo.
The relationships between such changes in sugar chains and diseases have also been gradually elucidated. For example, Patent Document 1 describes as follows with regard to prostate-specific antigens (hereinafter, referred to as “PSAs”) which indicate that a subject has a prostate disease. That is, it is described that, as compared to blood samples derived from prostatic hyperplasia patients, those blood samples derived from prostate cancer patients contain a greater amount of a prostate-specific antigen having a specific sugar residue, i.e., an N-acetyl-D-galactosamine β1-4 N-acetylglucosamine (hereinafter, referred to as “LacdiNAc”) residue (this prostate-specific antigen is hereinafter referred to as “LacdiNAc-PSA”) and/or a fucose-α(1,2)-galactose β1→4 N-acetylglucosamine residue in its sugar chain. This means that the onset of prostate cancer changes the sugar chains of PSAs and this leads to an increase in the amount of PSAs having the above-described specific sugar residue(s), as a result of which a high concentration of PSAs having the specific sugar residue(s) is observed in blood samples of prostate cancer patients. On the other hand, since the onset of prostatic hyperplasia does not cause such a change in the sugar chains, prostatic hyperplasia patients are not observed with a change in the concentration of PSAs having the specific sugar residue(s). On the basis of this, Patent Document 1 discloses a method of distinguishing prostate cancer by fraction measurement of sugar chains, which method is capable of distinguishing a prostate cancer patient from a prostatic hyperplasia patient by measuring, in their blood samples, the concentration of PSAs having the above-described specific sugar residue(s).
In addition, there have been known, for example, such methods of identifying liver cancer by fraction measurement of α-fetoprotein (AFP) sugar chain as disclosed in Patent Document 2 and Non-patent Document 1, as well as such methods of identifying adenocarcinoma by fraction measurement of carcinoembryonic antigen (CEA) sugar chain as disclosed in Non-patent Documents 2 and 3.
For specific detection of a glycoprotein containing a specific sugar residue in its sugar chain, proteins called lectin that are capable of specifically recognizing and binding to such sugar residue are widely utilized. This is because it is very difficult to prepare an antibody whose epitope is a sugar chain, particularly an antibody whose epitope is a specific sugar residue, and such an antibody is thus hardly available. Lectins not only are inexpensive and available in a large amount but also have excellent stability and can thus be stored over a long time.
For example, Wisteria floribunda lectin (Wisteria floribunda agglutinin: hereinafter, referred to as “WFA”) is known to have N-acetylgalactosamine as its primary binding sugar residue. Patent Document 1 discloses a method in which WFA having such a property is bound to a carrier and loaded to a column and a PSA having a LacdiNAc residue in a side chain of an asparagine-linked sugar chain is subsequently fractionated and quantified by ELISA or the like. In addition, Patent Document 3 discloses a method in which a solid-phase anti-PSA antibody and a fluorescently labeled WFA are allowed to form a sandwich complex with a PSA having a LacdiNAc residue in a side chain of its sugar chain and this PSA having the specific sugar residue is then quantified by SPFS (Surface Plasmon-field enhanced Fluorescence Spectroscopy).
Lectins, however, have such drawbacks of having lower binding activity and lower specificity of binding to sugar chains than antibodies. For example, Wisteria floribunda lectin (WFA) has N-acetylgalactosamine residue as its primary binding site; however, Wisteria floribunda lectin slightly binds with a galactose residue as well. Therefore, when a galactose residue-containing sugar chain exists in a reaction system, Wisteria floribunda lectin also binds to this sugar chain and thus cannot be specifically bound only to N-acetylgalactosamine residue-containing sugar chains.
Sandwich assays capable of simply and quantitatively analyzing a specific sugar chain-containing protein (glycoprotein) as a substance to be detected are performed using a lectin having such characteristics in combination with an antibody. An antibody which specifically binds to a protein moiety of a glycoprotein is immobilized on a substrate and used as a solid-phase antibody, and a lectin whose primary binding target is a sugar residue contained in a sugar chain of a glycoprotein is linked with a labeling agent and used as a labeled lectin.
However, although this technique is effective when the substance to be detected, which is a glycoprotein, is purified to a certain extent, since the labeled lectin binds not only to a sugar chain of the substance to be detected but also to sugar chains of contaminants, this technique generates a large background (noise) and shows markedly reduced performance in terms of sensitivity and quantitative capacity in a system that contains a large amount of contaminants, for example, glycoproteins and glycolipids other than the substance to be detected, as in blood, urine and the like that are used as a sample in ordinary disease diagnosis. Therefore, in ordinary diagnosis using blood or the like as a sample, it accompanies a great deal of difficulty to accurately perform a quantitative analysis by a sandwich assay using a lectin and an antibody and, in fact, such a sandwich assay is utilized only in diagnosis where the detection subject is a limited type of glycoprotein that is contained in serum at an extremely high concentration (about several μg/mL).
As methods for reducing the effects (background) of contaminants, there have been examined, for example, a method of using a blocking agent such as bovine serum albumin (BSA) or casein for inhibiting the adsorption of serum contaminants to the surface of a support on which an antibody is immobilized (Patent Document 4); a method of adding an adsorbent, for example, a polymer or a sugar chain complex such as glycosaminoglycan or heparin, for allowing contaminants (non-specific substances), which inhibit antigen-antibody reaction and cause noise, to adsorb thereto and thereby removing the contaminants from the reaction system (Patent Documents 5 and 6); and a method of using a washing liquid having a specific salt strength and a specific formation of surfactant and the like for efficiently removing contaminants adsorbed to a support (Patent Document 4).
However, even with the above-described background-reducing methods, it is an extremely rare case where a drastic effect is attained in a quantitative analysis based on sandwich assay using a lectin and an antibody, and there is also a problem that the search and examination of a blocking agent suitable for the subject of interest require tremendous man-hours.
Meanwhile, the present applicant previously proposed a method of detecting a substance to be detected (analyte) having a detection target sugar chain in a sample using a labeling lectin that binds to plural kinds of sugar chains including the detection target sugar chain and non-detection-target sugar chains, wherein the method comprises: a labeling treatment of bringing the labeling lectin into contact with the substance to be detected prior to the detection step of detecting the substance to be detected bound with the labeling lectin; and a masking treatment of bringing a sugar chain-recognizing molecule for masking, which binds to at least one of the non-detection-target sugar chains, into contact with contaminants having the non-detection-target sugar chains (Patent Document 7). That is, by performing the masking treatment on the non-detection-target sugar chains of contaminants to which the labeling lectin collaterally binds using the sugar chain-recognizing molecule for masking, the labeling lectin is enabled to preferentially bind to the detection target sugar chain contained in the substance to be detected, so that the background noise can be reduced and the detection sensitivity and quantitative performance for the substance to be detected can be improved. As the sugar chain-recognizing molecule for masking, a lectin which mainly recognizes and binds to the non-detection-target sugar chains rather than the detection target sugar chain and is different from the labeling lectin can be used.
However, the method of detecting a substance to be detected according to Patent Document 7 is applicable when a contaminant has a sugar chain containing a sugar residue that is recognized by the same lectin but different from that of the substance to be detected. Further, when such a contaminant is contained in a sample in a large amount, in the invention described in Patent Document 7, since it is required to add a sugar chain-recognizing molecule for masking (a lectin of a type that is different from the labeling lectin) at a high concentration, the reagent cost is increased or else the masking efficiency is reduced. Patent Document 7 discloses neither a means capable of efficiently suppressing the effect of such contaminant having a sugar chain that contains the same sugar residue as the substance to be detected, in a sample in a large amount, nor that the use of such a means can actually markedly improve the measurement sensitivity and quantitative performance for the substance to be detected.
Further, the present applicant has also proposed a method of suppressing non-specific signals in SPFS immunoassay in which a glycoprotein can be a compound to be measured, the method comprising performing at least one of the followings for the suppression of non-specific signals originating from contaminants: a pretreatment of adding an acid or an alkali to a sample; a pretreatment of adding a metal ion to the sample; and a pretreatment of heating the sample (Patent Document 8). By performing these pretreatments to change (modify) the structures of the contaminants, non-specific adsorption of the contaminants to a sensor section can be inhibited. However, in Patent Document 8 as well, there is disclosed no means capable of efficiently suppressing the effects of a contaminant (e.g., glycoprotein) having a sugar chain that contains the same sugar residue as the substance to be detected when such a contaminant is contained in a sample in a large amount.
In terms of these points, sandwich assay using a labeled lectin still has room for improvement.